Coacervate micro and/or nano droplets and hydrogels

ABSTRACT

A composition includes a plurality of coacervate micro and/or nanodroplets of oxidized alginate and a methacrylated gelatin.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application No.62/022,343, filed Jul. 9, 2014, the subject matter, which isincorporated herein by reference in its entirety.

BACKGROUND

Complex coacervation is known as liquid-liquid phase separation inaqueous solution by spontaneous aggregation associated withelectrostatic matching between two oppositely charged polyelectrolytes.Complex coacervates have been used for direct complexation betweenbioactive molecules and polysaccharides, micro- or nanoencapsulation ofbioactive molecules or cells, and surface coating of particles, due totheir unique physicochemical characteristics that can be easilymodulated by pH, ionic strength, charge density, and stoichiometry ofinteracting molecules. However, these systems often require cytotoxicsurfactants and/or expensive equipment. The ability to cheaply formcytocompatible coacervates under mild conditions that permit thecompartmentalized encapsulation of cells and bioactive factors viasimple mixing would be valuable for tissue engineering strategies as itwould allow for control over their spatial distribution. However, nocoacervate system has been reported capable of simultaneous cellencapsulation and the formation of drug-laden microdroplets underphysiological conditions that could be used as a three-dimensionalbiomaterial for cell encapsulation and transplantation, and tissueengineering applications, due to the harsh physicochemical conditions[i.e., low pH (<4) and high temperature (˜60° C.)] typically requiredfor complex coacervation formation.

SUMMARY

Embodiments described herein relate to a complex cytocompatiblecoacervate system that can be used to form coacervate micro and/ornanodroplets and/or coacevate-laden hydrogels (i.e., coacevatehydrogels). The coacervate micro and/or nanodroplets and/or hydrogelscan provide localized, sustained, and/or controlled release of bioactiveagents, such as polypeptides and polynucleotides, to cells in or aboutthe coacervate micro and/or nanodroplets and/or hydrogels underphysiological conditions in a spatial and/or temporally controlled orpredetermined manner.

In some embodiments, a composition can be provided that includes aplurality of coacervate micro and/or nanodroplets. The coacervate microand/or nanodroplets can include oxidized alginate, a methacrylatedgelatin. At least one bioactive agent can be incorporated in the microand/or nanodroplets. The oxidized alginate can have an oxidationpercentage up to about 50%, for example, about 1% to about 50% or about5% to about 25%. The oxidized alginate can also be methacrylated andhave a methacrylation percentage up to about 75%, for example, about 5%to about 45%. The methacrylated gelatin can have a methacrylationpercentage, up to about 100%, for example, about 5% to about 99%. Insome embodiments, the methacrylated gelatin can have a methacrylationpercentage of at least about 10%, for example, at least about 75%.

In some embodiment, the composition can include a cytocompatiblehydrogel matrix in which the coacervate micro and/or nanodroplets aresuspended. The hydrogel matrix can include cross-linked oxidizedmethacrylated alginate.

In still other embodiments, the composition can include a plurality ofcells that are suspended in the hydrogel matrix. The plurality ofcoacervate micro and/or nanodroplets can provide controlled release ofthe bioactive agent to the plurality of cells suspended or provided inthe matrix. For example, BMP-2 loaded coacervate micro and/ornanodroplets can be used to provide controlled release of BMP-2 toprogenitor cells, such as hMSCs, suspended in the hydrogel matrix.

Other embodiments relate to a hydrogel comprising a crosslinked oxidizedalginate and a methacrylated gelatin that form or provide a hydrogelmatrix and a plurality of coacervate micro and/or nanodroplets. Thehydrogel can include at least one bioactive agent incorporated in themicro and/or nanodroplets and/or matrix. The oxidized alginate can havean oxidation percentage up to about 50%, for example, about 1% to about50% or about 5% to about 25%. The oxidized alginate can also bemethacrylated and have a methacrylation percentage up to about 75%, forexample, about 5% to about 45%. The methacrylated gelatin can have amethacrylation percentage, up to about 100%, for example, about 5% toabout 99%. In some embodiments, the methacrylated gelatin can have amethacrylation percentage of at least about 10%, for example, at leastabout 75%. In some embodiments, a plurality of cells is incorporated orprovided in the hydrogel matrix and the plurality of coacervate microand/or nanodroplets can provide controlled release of the bioactiveagent to the plurality of cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates ¹H-NMR spectra of OMAs with various degrees ofoxidation and methacrylation in D₂O. The OMAs were dissolved in D₂O (2w/v %), and ¹H-NMR spectra were recorded on a Varian Unity-300 (300 MHz)NMR spectrometer (Varian Inc.) using 3-(trimethylsilyl)propionic acid-d₄sodium salt (0.05 w/v %) as an internal standard.

FIG. 2 illustrates ¹H-NMR spectra of gelatins and GelMAs with variousdegrees of methacrylation in D₂O. The GelMAs were dissolved in D₂O (2w/v %), and ¹H-NMR spectra were recorded on a Varian Unity-300 (300 MHz)NMR spectrometer (Varian Inc.) using 3-(trimethylsilyl)propionic acid-d₄sodium salt (0.05 w/v %) as an internal standard.

FIGS. 3(A-W) illustrate images and graphs showing characterization ofOMA/GelMA coacervates. (A-B), OMA/GelMA coacervate microdroplets formedby 20 w/v % OMA with various degrees of oxidation and methacrylation and(A) 20 w/v % H-GelMA-A or (B) 20 w/v % H-GelMA-B in PBS at pH 7.4. (C)Photocrosslinked OMA/H-GelMA-B coacervate microdroplet-laden hydrogels.The scale bars indicate 100 μm. Size distribution of OMA/GelMAcoacervate microdroplets formed with (d) 10OX15MA/H-GelMA-A, (E)10OX25MA/H-GelMA-A, (F) 10OX45MA/H-GelMA-A, (G) 17.5OX15MA/H-GelMA-A,(H) 17.5OX25MA/H-GelMA-A, (I) 17.5OX45MA/H-GelMA-A, (J)25OX15MA/H-GelMA-A, (K) 25OX25MA/H-GelMA-A, (L) 25OX45MA/H-GelMA-A, (M)10OX15MA/H-GelMA-B, (N) 10OX25MA/H-GelMA-B, (O) 10OX45MA/H-GelMA-B, (P)17.5OX15MA/H-GelMA-B, (Q) 17.5OX25MA/H-GelMA-B, (R)17.5OX45MA/H-GelMA-B, (S) 25OX15MA/H-GelMA-B, (T) 25OX25MA/H-GelMA-B and(U) 25OX45MA/H-GelMA-B. The coacervate microdroplet diameters weremeasured using NIH ImageJ ananlysis software (n>400 per group). Elasticmoduli in compression of OMA/GelMA coacervate microdroplet-ladenhydrogels formed with (V) H-GleMA-A and (W) H-GelMA-B.

FIGS. 4(A-F) illustrate graphs showing turbidity of (A) OAs andgelatins, (B) OMAs and gelatins, (C) OAs and L-GelMAs, (D) OAs andH-GelMAs, (E) OMAs and L-GelMAs, and (F) OA and H-GelMA-A/gelatin-Abefore and after mixing by absorbance measurement at 500 nm to evaluatethe degrees of complex coacervate formation.

FIG. 5 illustrates a plot showing release profiles of BMP-2 fromphotocrosslinked heparin-modified OMA/BMP-2 in GelMA (black circle) andBMP-2 in OMA/GelMA (red triangle) coacervate hydrogels (N=5).

FIGS. 6(A-B) illustrate (A) Representative live/dead images ofencapsulated hMSCs in photocrosslinked OMA/GelMA, OMA/BMP-2 in GelMA,and BMP-2 in OMA/GelMA coacervate hydrogels at days 0, 7, 14, 21 and 28.Photoencapsulated hMSCs in the coacervate hydrogels were cultured inosteogenic differentiation media at 37° C. with 5% CO₂. The scale barsindicate 100 μm. (B) A graph showing quantification of DNA in theconstructs at days 7, 14, 21, 28, 42, 56 and 112. *p<0.05 compared withBMP-2 in the hydrogel groups at a specific time point.

FIG. 7 illustrates a graph showing quantification of calcium content(N=6) in the heparin-modified constructs. All quantitative data isexpressed as mean±standard deviation. Statistical analysis was performedwith one-way analysis of variance (ANOVA) with Tukey significantdifference post hoc test using Origin software. *p<0.05 compared withBMP-2 in OMA/GelMA and OMA/BMP-2 in GelMA groups at a specific timepoint.

FIGS. 8(A-H) illustrate the formation of OMA/GelMA coacervates. (A-B),Schematic illustrations of preparation and chemical structures of (A)OMA and (B) GelMA. (C) Schematic illustration of Schiff base reactionbetween the aldehyde group of the OMA and amine group of the GelMA.(D-F), Representative optical photomicrographs of OMA/GelMA coacervatemicrodroplets formed by (D) 17.5OX15MA and H-GelMA-B, (E) 17.5OX25MA andH-GelMA-B, and (F) 17.5OX45MA and H-GelMA-B. The scale bars indicate 100μm. (G) Turbidity of OMA/GelMA solutions prepared at pH 7.4 before andafter mixing of two solutions by the measurement of the absorbance at500 nm to evaluate the degrees of complex coacervate formation. (H)Turbidity of OMA (25OX45MA)/H-GelMA coacervate as a function of pH. Allquantitative data is expressed as mean±standard deviation (N=3).Statistical analysis was performed with one-way analysis of variance(ANOVA) with Tukey significant difference post hoc test using Originsoftware (OriginLab Co). The absorbance of all groups significantlyincreased after mixing (p<0.001). *p<0.05 compared with H-GelMA-B at aspecific pH.

FIGS. 9(A-I) illustrate microstructural characterization of OMA/GelMAcoacervates. (A-B), Schematic illustrations for synthesis offluorescently-labeled (A) 17.5OX15MA and (B) H-GelMA-A. (C-E),Representative fluorescence photomicrographs of OMA/GelMA coacervates.(C) Red channel, (D) blue channel and (E) merged image. (F-H),Representative fluorescence photomicrographs of individual OMA/GelMAcoacervate microdroplet with high magnification. (F) Red channel, (G)blue channel and (H) merged image. The scale bars indicate 50 μm. (I)Schematic representation of the formation of OMA/GelMA coacervatemicrodroplets.

FIGS. 10(A-L) illustrate the effect of alginate and gelatinmethacrylation on coacervate formation. (A-F), Representative photographof (A) OA/Gelatin, (B) OMA/Gelatin, (C) OA/L-GelMA, (D) OA/H-GelMA, (E)OMA/L-GelMA and (F) OMA/H-GelMA mixtures in a 96-well plate. (G-H),Schematic microstructure and representative fluorescence images [redchannel (top), blue channel (middle), and merged image (bottom)] of (G)OA/Gelatin, (H) OMA/Gelatin, (I) OA/L-GelMA, (J) OA/H-GelMA, (K)OMA/L-GelMA and (L) OMA/H-GelMA mixtures. The scale bars indicate 30 μm.

FIGS. 11(A-K) illustrate phothoencapsulation of hMSCs and BMP-2 inOMA/GelMA coacervate hydrogels induces hMSC osteogenesis. (A) Schematicillustration of in situ formation of BMP-2-loaded coacervatemicrodroplets-embedded hydrogel for osteogenic differentiation ofphotoencapsulated hMSC. (B) Release profiles of BMP-2 fromphotocrosslinked OMA/BMP-2 in GelMA and BMP-2 in OMA/GelMA coacervatemicrodroplet-laden hydrogels (N=5). (C-E), Live/Dead staining ofencapsulated hMSCs in photocrosslinked (C) OMA/GelMA, (D) OMA/BMP-2 inGelMA and (E) BMP-2 in OMA/GelMA coacervate microdroplet-laden hydrogelsafter 28 days culture in osteogenic differentiation media. (F-H),Quantification of (F) ALP/DNA (N=6), and (G) relative Runx2 (N=6) and(h) BSP (N=6) gene expression in hMSCs encapsulated within hydrogels.(I-K), Mineralization of cell-hydrogel constructs analyzed by (I and J)Alizarin red staining and (J) quantification of calcium content (N=6) inthe constructs. The scale bars indicate 100 μm. All quantitative data isexpressed as mean±standard deviation. Statistical analysis was performedwith one-way analysis of variance (ANOVA) with Tukey significantdifference post hoc test using Origin software. *p<0.05 compared withBMP-2 in OMA/GelMA group at a specific time point. **p<0.05 comparedwith OMA/GelMA group at a specific time point.

DETAILED DESCRIPTION

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises, such as Current Protocolsin Molecular Biology, ed. Ausubel et al., Greene Publishing andWiley-Interscience, New York, 1992 (with periodic updates). Unlessotherwise defined, all technical terms used herein have the same meaningas commonly understood by one of ordinary skill in the art to which thepresent invention pertains. Commonly understood definitions of molecularbiology terms can be found in, for example, Rieger et al., Glossary ofGenetics: Classical and Molecular, 5th Edition, Springer-Verlag: NewYork, 1991, and Lewin, Genes V, Oxford University Press: New York, 1994.The definitions provided herein are to facilitate understanding ofcertain terms used frequently herein and are not meant to limit thescope of the present invention.

The term “antisense” nucleic acid refers to oligonucleotides whichspecifically hybridize (e.g., bind) under cellular conditions with agene sequence, such as at the cellular mRNA and/or genomic DNA level, soas to inhibit expression of that gene, e.g., by inhibiting transcriptionand/or translation. The binding may be by conventional base paircomplementarily, or, for example, in the case of binding to DNAduplexes, through specific interactions in the major groove of thedouble helix.

The term “bioactive agent” refers to any agent capable of promotingtissue formation, destruction, and/or targeting a specific disease state(e.g., cancer). When administered to a host, both human and animal,e.g., the bioactive agent may be used as part of a prophylatic ortherapeutic treatment. Examples of bioactive agents can include, but arenot limited to, chemotactic agents, various proteins (e.g., short termpeptides, bone morphogenic proteins, collagen, glycoproteins, andlipoprotein), cell attachment mediators, biologically active ligands,integrin binding sequence, various growth and/or differentiation agentsand fragments thereof (e.g., epidermal growth factor (EGF), hepatocytegrowth factor (HGF), vascular endothelial growth factors (VEGF),fibroblast growth factors (e.g., bFGF), platelet derived growth factors(PDGF), insulin-like growth factor (e.g., IGF-I, IGF-II) andtransforming growth factors (e.g., TGF-β I-III)), parathyroid hormone,parathyroid hormone related peptide, bone morphogenic proteins (e.g.,BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-13, BMP-14), transcriptionfactors, such as sonic hedgehog, growth differentiation factors (e.g.,GDF5, GDF6, GDF8), recombinant human growth factors (e.g., MP52 and theMP-52 variant rhGDF-5), cartilage-derived morphogenic proteins (CDMP-1,CDMP-2, CDMP-3), small molecules that affect the upregulation ofspecific growth factors, tenascin-C, hyaluronic acid, chondroitinsulfate, fibronectin, decorin, thromboelastin, thrombin-derivedpeptides, heparin-binding domains, heparin, heparan sulfate,polynucleotides, DNA fragments, DNA plasmids, MMPs, TIMPs, interferingRNA molecules, such as siRNAs, oligonucleotides, proteoglycans,glycoproteins, glycosaminoglycans, and DNA encoding for shRNA. Inaddition, biological entities, such as viruses, virenos, and prions areconsidered bioactive agents. The bioactive agents may be water-solubleor water-insoluble and may include those having a high molecular weight,such as proteins, peptides, carbohydrates and glycoproteins.

The term “biocompatibility” or “biocompatible” when used in relation tocoacervates described herein refers to coacervates that are neitherthemselves toxic to the host (e.g., an animal or human), nor degrade (ifat all) at a rate that produces byproducts at toxic concentrations inthe host. To determine whether any subject coacervates arebiocompatible, it may be necessary to conduct a toxicity analysis. Suchassays are well known in the art.

The term “biodegradable” refers to those embodiments in whichcoacervates or hydrogels described herein are intended to degrade duringuse. In general, degradation attributable to biodegradability involvesthe degradation of a coacervate or hydrogel into its constituents andencapsulated materials. The degradation rate of a biodegradablecoacervate or hydrogel often depends in part on a variety of factors,including the identity of any constituents that form the coacervate andhydrogel and their ratio, the identity and loading of any material(including bioactive agent encapsulated in a coacervate), how anycoacervate may be crosslinked and to what extent. For example, acoacervate that is crosslinked will, in all likelihood, degrade moreslowly than one that is not crosslinked.

The term “cell” can refer to any progenitor cell, such as totipotentstem cells, pluripotent stem cells, and multipotent stem cells, as wellas any of their lineage descendant cells, including more differentiatedcells. The terms “stem cell” and “progenitor cell” are usedinterchangeably herein. The cells can derive from embryonic, fetal, oradult tissues. Exemplary progenitor cells can be selected from, but notrestricted to, totipotent stem cells, pluripotent stem cells,multipotent stem cells, mesenchymal stem cells (MSCs), hematopoieticstem cells, neuronal stem cells, hematopoietic stem cells, pancreaticstem cells, cardiac stem cells, embryonic stem cells, embryonic germcells, neural crest stem cells, kidney stem cells, hepatic stem cells,lung stem cells, hemangioblast cells, and endothelial progenitor cells.Additional exemplary progenitor cells are selected from, but notrestricted to, de-differentiated chondrogenic cells, chondrogenic cells,cord blood stem cells, multi-potent adult progenitor cells, myogeniccells, osteogenic cells, tendogenic cells, ligamentogenic cells,adipogenic cells, and dermatogenic cells.

When used with respect to the bioactive agent, the term “controlledrelease” is intended to mean that the bioactive agent is released overtime in contrast to a bolus type administration in which the entireamount of the bioactive agent is presented to the target at one time.The release will vary as explained below.

The term “gene” or “recombinant gene” refers to a nucleic acidcomprising an open reading frame encoding a polypeptide, including bothexonic and (optionally) intronic sequences.

The term “gene construct” refers to a vector, plasmid, viral genome orthe like which includes an “coding sequence” for a polypeptide or whichis otherwise transcribable to a biologically active RNA (e.g.,antisense, decoy, ribozyme, etc), can transfect cells, preferablymammalian cells, and can cause expression of the coding sequence incells transfected with the construct. The gene construct may include oneor more regulatory elements operably linked to the coding sequence, aswell as intronic sequences, poly adenylation sites, origins ofreplication, marker genes, etc.

The term “host cell” or “target cell” refers to a cell transduced with aspecified transfer vector. The cell is optionally selected from in vitrocells such as those derived from cell culture, ex vivo cells, such asthose derived from an organism, and in vivo cells, such as those in anorganism.

The term “incorporated” or “encapsulation,” when used in reference to abioactive agent or other material and a coacervate, denotes formulatinga bioactive agent or other material into a coacervate useful forcontrolled release of such agent or material. As used herein, thoseterms contemplate any manner by which a bioactive agent is incorporatedinto a coacervate, including for example: distributed throughout thematrix, appended to the surface of microdroplets, and encapsulatedinside the matrix or microdroplets. The term “coincorporation” or“coencapsulation” as used herein refers to the incorporation of abioactive agent in a coacervate and at least another bioactive agent orother material.

The term “microspheres”, “microdroplets”, “nanospheres”, “nanodroplets”,or “micro and/or nanodroplets” are used interchangeably and refer tosubstantially spherical structures formed by a coacervation process. Themicro and/or nanodroplets generally have a matrix-type structure, andcan incorporate and/or encapsulate a bioactive agent within the matrix.The micro and/or nanodroplets generally have a size distribution withinthe range of from about 10 nM to about 100 μM. In certain embodiments,over 90% of the microdroplets formed in a single preparation ofcoacervates have a diameter in excess of about 5 μM. Other sizes arealso contemplated herein

When a large number of micro and/or nanodroplets are formed in acoacervate composition, they may have a variable size. In certainembodiments, the size distribution may be uniform, e.g., within lessthan about a 20% standard deviation of the median volume diameter, andin other embodiments, still more uniform or within about 10% of themedian volume diameter.

The term “modulation” refers to both up regulation (i.e., activation orstimulation) and down regulation (i.e., inhibition or suppression) of aresponse.

The term “nucleic acid” refers to polynucleotides, such asdeoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single-stranded (such assense or antisense) and double-stranded polynucleotides. Exemplarynucleic acids for use in the subject invention include antisense, decoymolecules, recombinant genes (including transgenes) and the like.

The phrases “parenteral administration” and “administered parenterally”means modes of administration other than enteral and topicaladministration, usually by injection, and includes, without limitation,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intra-articulare, subcapsular, subarachnoid,intraspinal and intrasternal injection and infusion.

The phrase “pharmaceutically acceptable” refers to those coacervates anddosages thereof within the scope of sound medical judgment, suitable foruse in contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The term “prophylactic or therapeutic” treatment refers toadministration to the host of the subject micro and/or nanodroplets. Ifit is administered prior to clinical manifestation of the unwantedcondition (e.g., disease or other unwanted state of the host animal)then the treatment is prophylactic, i.e., it protects the host againstdeveloping the unwanted condition, whereas if administered aftermanifestation of the unwanted condition, the treatment is therapeutic(i.e., it is intended to diminish or ameliorate the existing unwantedcondition or side effects therefrom).

The terms “protein,” “polypeptide” and “peptide” are usedinterchangeably when referring to a gene product.

“Recombinant host cells” refers to cells which have been transformed ortransfected with vectors constructed using recombinant DNA techniques.

The terms “recombinant protein,” “heterologous protein” and “exogenousprotein” are used interchangeably to refer to a polypeptide which isproduced by recombinant DNA techniques, wherein generally, DNA encodingthe polypeptide is inserted into a suitable expression vector which isin turn used to transform a host cell to produce the heterologousprotein. That is, the polypeptide is expressed from a heterologousnucleic acid.

As used herein, the term “subject” can refer to any animal, including,but not limited to, humans and non-human animals (e.g., rodents,arthropods, insects, fish (e.g., zebrafish), non-human primates, ovines,bovines, ruminants, lagomorphs, porcines, caprines, equines, canines,felines, ayes, etc.), which is to be the recipient of a particulartreatment. Typically, the terms “patient” and “subject” are usedinterchangeably herein in reference to a human subject.

The phrases “systemic administration,” “administered systematically,”“peripheral administration” and “administered peripherally” mean theadministration of a subject supplement, composition, therapeutic orother material such that it enters the patient's system and, thus, issubject to metabolism and other like processes, for example,subcutaneous administration.

The phrase “therapeutically effective amount” means that amount of abioactive agent that, when present in a coacervate, produces somedesired effect at a reasonable benefit/risk ratio applicable to anymedical treatment. In certain embodiments, a therapeutically effectiveamount of a bioactive agent for in vivo use will likely depend on anumber of factors, including: the rate of release of the bioactive agentfrom the coacervate, which will depend in part on the chemical andphysical characteristics of the such coacervate, the identity of thebioactive agent, the mode and method of administration; any othermaterials incorporated in the coacervate in addition to the bioactiveagent.

The term “treating” as used herein is intended to encompass curing aswell as ameliorating at least one symptom of any condition or disease.

Embodiments described herein relate to a complex cytocompatiblecoacervate system that can be used to form coacervate micro and/ornanodroplets and/or coacervate-laden hydrogels (i.e., coacervatehydrogels). The coacervate micro and/or nanodroplets and/or hydrogelscan provide localized, sustained, and/or controlled release of bioactiveagents, such as polypeptides and polynucleotides, to cells in or aboutthe coacervate micro and/or nanodroplets and/or hydrogels underphysiological conditions in a spatial and/or temporally controlled orpredetermined manner.

It was found that coacervate micro and/or nanodroplets and/orcoacervate-laden hydrogels can be formed under physiological conditionsfrom the simple mixing of photocrosslinkable oxidized alginate (OA) oroxidized, methacrylated alginate (OMA) with methacrylated gelatin(GelMA) at a wide pH range and room temperature. This system enablessimultaneous creation of bioactive-laden micro and/or nanodroplets andencapsulation of cells in photopolymerized coacervate hydrogels underphysiological conditions. The coacervate system can be utilized as aplatform for in situ formation of three-dimensional biomaterials forcell encapsulation and transplantation as well as localized, sustained,controlled, and/or spatial bioactive agent delivery to cells and/ortissue, such as encapsulated cells, for therapeutic applications andtissue engineering applications. The ability to readily formcytocompatible coacervates under mild conditions that permit thecompartmentalized encapsulation of cells and/or bioactive agents viasimple mixing can be valuable for tissue engineering strategies as itallows for control over their spatial distribution.

In some embodiments, the complex coacervate system described herein caninclude an oxidized alginate or oxidized methacrylated alginate thatwhen mixed with a methacrylated gelatin forms a plurality of coacervatemicro and/or nanodroplets under physiological pH and temperature. Theoxidized alginate can be formed by periodate oxidation of alginateusing, for example, sodium periodate. Periodate oxidation can cleave thecarbon-carbon bond of the cis-diol group in the uronate residue ofalginate and alter the chain conformation. The alignate can be oxidizedusing a periodate to provide an alginate oxidation up to about 50%, forexample, about 5% to about 50% or about 10% to about 25%. The oxidizedalginate can optionally then be methacrylated by reacting the oxidizedalginate with a methacrylate reactant, such as 2-aminoethylmethacrylate, to provide an alginate methacrylation up to about 75%, forexample, about 5% to about 45% or about 15% to about 45%. In someembodiment, the oxidized methacrylated alginate can have an alginateoxidation percentage of about 10% to about 25% and an alginatemethacrylation percentage of about 10% to about 25%.

The methacrylated gelatin can be prepared by reacting gelatin withmaleic anhydride. The gelatin methacrylation percentage can be varied upto about 100%, for example, about 5% to about 99%. Advantageously, thegelatin methacrylation percentage can be at least about 10% (e.g., atabout 75%) to enhance formation of the coacervate upon mixing of theoxidized methacrylated alginate and the methacrylated gelatin.

Upon mixing, the methacrylated gelatin can form imine bond-basedcovalent complexes with the oxidized alginate or oxidized methacrylatedalginate and a plurality of coacervate micro and/or nanodroplets thatare suspended in a coacervate liquid phase or matrix. The coacervatemicro and/or nanodroplets can be substantially and/or uniformlyspherical and be distributed substantially uniformly throughoutcoacervate.

The coacervate micro and/or nanodroplets can be primarily orsubstantially composed of the methacrylated gelatin with the oxidizedalginate or oxidized methacrylated alginate being provided on themicrodroplet surface shell and in the surrounding equilibrium phase ormatrix of the coacervate. Since methacrylate groups are hydrophobic,methacrylation of alginate and gelatin can increase the hydrophobicity.Because gelatin tends to aggregate by hydrophobic interactions, which isfurther enhanced by its methacrylation, the methacrylated gelatin canmore rapidly aggregate and form coacervate mcrodroplets within a fewseconds.

In some embodiment, the liquid phase surrounding the coacervate microand/or nanodroplets can be cross-linked so a hydrogel is formed with aplurality of the coacervate micro and/or nanodroplets suspended in amatrix of the hydrogel. Such crosslinks may be between the same ordifferent constituents of the coacervate, and may involve bioactiveagents or other materials incorporated therein. There are a number ofagents and methods of using the foregoing that may be used to affectsuch crosslinking. In one embodiment, crosslinking may be affected byuse of including a constituent in the coacervate that isphotocrosslinkable. For example, the oxidized alginate or oxidizedmethacrylated alginate can be photocrosslinkable with UV light in thepresence of photoinitiators to form a photocrosslinked hydrogel. Thephotoinitiator can include any photoinitiator that can initiate orinduce polymerization or crosslinking of a constituent, such as theoxidized methacrylated alginate, of the coacervate. Examples ofphotoinitiators can include 2-hydroxy,-4′-(2-hydroxyethoxy)-2-methylpropiophenone, camphorquinone, benzoinmethyl ether, 2-hydroxy-2-methyl-1-phenyl-1-propanone, diphenyl(2,4,6-trimethylbenzoyl) phosphine oxide, benzoin ethyl ether,benzophenone, 9, 10-anthraquinone, ethyl-4-N,N-dimethylaminobenzoate,diphenyliodonium chloride and derivatives thereof. Other examples arepresented in U.S. Pat. No. 5,858,746, which is herein incorporated byreference in its entirety. In addition, any photocrosslinkableconstituent of a coacervate may be used as a primer or coupling agent tomodify the exterior of the coacervate. For example, such primer orcoupling agent may be used to react to enhance biocompatibility and toincrease adhesion to cells, cell aggregates, tissues and syntheticmaterials.

The at least on bioactive agent can be incorporated and/or encapsulatedin the coacervate micro and/or nanodroplets to provide localized,sustained, and/or controlled release of the at least one bioactiveagents to cells in or about the coacervate micro and/or nanodropletsand/or hydrogels under physiological conditions in a spatial and/ortemporally controlled or predetermined manner By incorporating and/orencapsulating bioactive agent in a coacervate microdroplet, it ispossible, in certain embodiments, to provide a steady dosage of suchbioactive agent through a sustained or controlled release process. Inaddition, such encapsulation may protect the bioactive agent, or othermaterials from undesirable immunogenic, proteolytic or other events thatwould reduce the efficacy of the bioactive agent. It will be appreciatethat the at least one bioactive agent can alternatively or additionallybe provided in the hydrogel matrix surrounding the micro and/ornanodroplets to further modify the release of the bioactive agent tocells in or about the micro and/or nanodroplets and/or hydrogel.

The at least one bioactive agent can include polynucleotides and/orpolypeptides encoding or comprising, for example, transcription factors,differentiation factors, growth factors, and combinations thereof. Theat least one bioactive agent can also include any agent capable ofpromoting tissue formation (e.g., bone and/or cartilage), destruction,and/or targeting a specific disease state (e.g., cancer). Examples ofbioactive agents include chemotactic agents, various proteins (e.g.,short term peptides, bone morphogenic proteins, collagen, glycoproteins,and lipoprotein), cell attachment mediators, biologically activeligands, integrin binding sequence, various growth and/ordifferentiation agents and fragments thereof (e.g., EGF), HGF, VEGF,fibroblast growth factors (e.g., bFGF), PDGF, insulin-like growth factor(e.g., IGF-I, IGF-II) and transforming growth factors (e.g., TGF-βI-III), parathyroid hormone, parathyroid hormone related peptide, bonemorphogenic proteins (e.g., BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-13,BMP-14), sonic hedgehog, growth differentiation factors (e.g., GDF5,GDF6, GDF8), recombinant human growth factors (e.g., MP-52 and the MP-52variant rhGDF-5), cartilage-derived morphogenic proteins (CDMP-1,CDMP-2, CDMP-3), small molecules that affect the upregulation ofspecific growth factors, tenascin-C, hyaluronic acid, chondroitinsulfate, fibronectin, decorin, thromboelastin, thrombin-derivedpeptides, heparin-binding domains, heparin, heparan sulfate,polynucleotides, DNA fragments, DNA plasmids, MMPs, TIMPs, interferingRNA molecules, such as siRNAs, DNA encoding for an shRNA of interest,oligonucleotides, proteoglycans, glycoproteins, and glycosaminoglycans.

The bioactive agents can be loaded, incorporated, and/or encapsulatedinto coacervate micro and/or nanodroplets and/or hydrogel during theirpreparation. For example, the bioactive agent can be initially combinedwith the methacrylated gelatin prior to mixing with the oxidizedmethacrylated alginate so that the bioactive agent is provided in thecoacervate micro and/or nanodroplets. Alternatively or additionally, theat least on bioactive agent can be combined with the oxidizedmethacrylated alginate prior to mixing with the methacrylated gelatin sothat the bioactive agent is provided in the surrounding matrix of thehydrogel. The amount of bioactive agent provided in the coacervate microand/or nanodroplets and/or hydrogel will depend on a number of factors,including: (i) the identity of the bioactive agent; (ii) thecoacervate's intended use, including any desired therapeutic effect forin vivo use; (iii) the chemical and physical properties of thecoacervate, including the release rate of encapsulated bioactive agentor other material under different conditions.

In certain embodiments, a sufficient amount of the bioactive agent canbe incorporated into the coacervate micro and/or nanodroplets and/orhydrogel to produce a therapeutically beneficial result. In thoseembodiments in which the bioactive agent is a polypeptide, such asBMP-2, the polypeptide loaded in any coacervate may range from less thanabout 0.05 to more than about 50 weight percent, or about 0.1, 0.25,0.5, 0.75, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 15, 20,25, 30, 35, 40, or 45 weight percent.

In addition to bioactive agents, other materials may be incorporatedinto a coacervate micro and/or nanodroplets and/or hydrogels. Suchadditional materials may affect the therapeutic and othercharacteristics of the coacervate that results. One example of suchanother material is an adjuvant. (Such materials may also be termedbioactive agents if appropriate.)

Alternatively, materials that augment the therapeutic effect of thebioactive agent may be incorporated into the coacervate micro and/ornanodroplets and/or hydrogel. For example, natural polymers, such asheparin, that control and/or delay the release of the bioactive agentcan be provided in the coacervate. (Such materials may also be referredto as bioactive agents as appropriate). The amount of any suchaugmenting agent to be loaded into any coacervate will depend on avariety of factors, including the nature of the such agent, thecoacervate, whether there are any other materials incorporated inaddition to the bioactive agent, and the like. For any such agent, thepresent invention contemplates incorporating a sufficient amount toaugment the therapeutic effect of the bioactive agent. In otherembodiments, the amount of such augmenting agent may range from about0.005% up to about 25%, or alternatively 0.01, 0.05, 0.1, 0.25, 0.5,1.0, 2.5, 5.0, 10, 15 or 20%.

In some embodiments, more than two different bioactive agents can beloaded into the coacervate micro and/or nanodroplets and/or hydrogel. Incertain embodiments, three, four, five or more bioactive agentsaugmenting agents, fillers or other materials may be incorporated in anycoacervate microdroplet and/or hydrogel.

The release rate of the bioactive agent in the coacervate micro and/ornanodroplets and/or hydrogel will vary with different embodiments. Forexample, one subject formulation may require at least an hour to releasea major portion of the bioactive agent into the surrounding medium,whereas another formulation may require about 1-24 hours, or even muchlonger. In certain embodiments, such release may result in release(over, say 1 to about 2,000 hours, or alternatively about 2 to about 800hours) of the bioactive agent or other material encapsulated in thecoacervate micro and/or nanodroplets. In certain embodiments, suchsubstance or other material may be released in an amount sufficient toproduce a therapeutically beneficial response.

The release profile of any bioactive agent or other material from thecoacervate micro and/or nanodroplets and/or hydrogel may vary indifferent embodiments. In one embodiment, the bioactive agent or othermaterial is released from the coacervate micro and/or nanodropletsand/or hydrogel in a pulsatile manner For example, such a pulsatilemanner may involve release of the bioactive agent or other material inthree phases: an initial burst, a slow release, and a second burst. Inanother embodiment of the present invention, the bioactive agent orother material is released in a sustained manner In still otherembodiments, a significant portion of the bioactive agent or othermaterial is released in an initial phase. In still other embodiments,the release profile is bi-phasic or multi-phasic.

In other embodiment, the coacervate hydrogel can be biodegradable and/orcytocompatible and include at least one cell dispersed on or within thehydrogel. The plurality of coacervate micro and/or nanodroplets in thehydrogel can provide controlled release of the bioactive agent to the atleast one cell provided in the hydrogel. For example, a plurality ofcells can be entirely or partly encapsulated within the coacervatehydrogel and the bioactive agent can be controllably release in aspatial or temporal manner to facilitate proliferation, growth, and/ordifferentiation of the cells. Cells can include any progenitor cell,such as a totipotent stem cell, a pluripotent stem cell, or amultipotent stem cell, as well as any of their lineage descendant cells,including more differentiated cells (described above), such as MSCs.

The cells can be autologous, xenogeneic, allogeneic, and/or syngeneic.Where the cells are not autologous, it may be desirable to administerimmunosuppressive agents in order to minimize immunorejection. The cellsemployed may be primary cells, expanded cells, or cell lines, and may bedividing or non-dividing cells. Cells may be expanded ex vivo prior tointroduction into or onto the hydrogel. For example, autologous cellscan be expanded in this manner if a sufficient number of viable cellscannot be harvested from the host subject. Alternatively oradditionally, the cells may be pieces of tissue, including tissue thathas some internal structure. The cells may be primary tissue explantsand preparations thereof, cell lines (including transformed cells), orhost cells.

Generally, cells can be introduced into the coacervate hydrogel invitro, although in vivo seeding approaches can optionally oradditionally be employed. In some embodiments, the cells may be mixedwith the photocrosslinkable oxidized methacrylated alginate prior tomixing with methacrylated gelatin/bioactive agent solution and formingthe coacervate. After mixing the cells with the photocrosslinkableoxidized methacrylated alginate, the cell/oxidized methacrylatedalginate solution can be mixed with the methacrylated gelatin/bioactiveagent solution so that a coacervate is formed with the cells beingprovided in alginate solution surrounding the coacervate micro and/ornanodroplets. The coacervate can then be crosslinked to form abiodegradable hydrogel with the cells being provided in the hydrogelmatrix and at least one bioactive agent being encapsulated in coacervatemicro and/or nanodroplets.

If the biodegradable coacervate hydrogel is to be implanted for use invivo after in vitro seeding, for example, sufficient growth medium maybe supplied to ensure cell viability during in vitro culture prior to invivo application. Once the coacervate hydrogel has been implanted, thenutritional requirements of the cells can be met by the circulatingfluids of the host subject.

In other embodiments, cells may be injected into the coacervate hydrogel(e.g., in combination with growth medium) or may be introduced by othermeans, such as pressure, vacuum, osmosis, or manual mixing.Alternatively or additionally, cells may be layered on the coacervatehydrogel, or the coacervate hydrogel may be dipped into a cellsuspension and allowed to remain there under conditions and for a timesufficient for the cells to incorporate within or attach to thecoacervate hydrogel. Generally, it is desirable to avoid excessivemanual manipulation of the cells in order to minimize cell death duringthe impregnation procedure. For example, in some situations it may notbe desirable to manually mix or knead the cells with the biodegradablecoacervate hydrogel; however, such an approach may be useful in thosecases in which a sufficient number of cells will survive the procedure.Cells can also be introduced into the coacervate hydrogel in vivo simplyby placing the hydrogel in the subject adjacent a source of desiredcells. Bioactive agents released from the biodegradable hydrogel mayalso recruit local cells, cells in the circulation, or cells at adistance from the implantation or injection site.

As those of ordinary skill in the art will appreciate, the number ofcells to be introduced into the biodegradable coacervate hydrogel willvary based on the intended application of the hydrogel and on the typeof cell used. Where dividing autologous cells are being introduced byinjection or mixing into the biodegradable coacervate hydrogel, forexample, a lower number of cells can be used. Alternatively, wherenon-dividing cells are being introduced by injection or mixing into thebiodegradable coacervate hydrogel, a larger number of cells may berequired.

In another embodiment, coacervate micro and/or nanodroplets and/orhydrogels may contain particles useful to locate the coacervate fordiagnostic applications and the like. In certain embodiments, coacervatemicro and/or nanodroplets and/or hydrogels may contain paramagnetic,superparamagnetic or ferromagnetic substances which are of use inmagnetic resonance imaging (MRI) diagnostics. For example, submicronparticles of iron or a magnetic iron oxide may be incorporated intocoacervate micro and/or nanodroplets and/or hydrogels to provideferromagnetic or superparamagnetic particles. Paramagnetic MRI contrastagents principally comprise paramagnetic metal ions, such as gadoliniumions, held by a chelating agent which prevents their release (and thussubstantially reduces their toxicity). In another embodiment, coacervatemicro and/or nanodroplets and/or hydrogels may contain submicronparticles, such as magnetic iron oxide, which permit the magneticseparation of coacervates. Other labeled compounds, such asradionucleides, e.g., ³H, ¹⁴C, ¹⁸F, ³²P, ^(99m)Tc, and ¹²⁵I, may also beutilized for visualizing cells and tissues, to which coacervate microand/or nanodroplets and/or hydrogels may be bound, by means of X-rays ormagnetic resonance imaging. Coacervate micro and/or nanodroplets and/orhydrogels may also contain in certain embodiments, ultrasound contrastagents, such as heavy materials, e.g., barium sulphate or iodinatedcompounds, to provide ultrasound contrast media.

In still other embodiments, the coacervate micro and/or nanodropletsand/or hydrogels may be conjugated to targeting molecules attached tothe surface of the coacervate microdroplet and/or hydrogel, such asmonoclonal antibodies that preferentially bind to a receptor or othersite of interest. In certain embodiments, such targeting may achievetargeted delivery in vivo of the micro and/or nanodroplets and/orhydrogel. To attach targeting molecules to the surface of any coacervatemicro and/or nanodroplets and/or hydrogels, it may be necessary toprovide coacervates linker molecules. Such linker molecules may be usedto attach targeting molecules. Alternatively, the constituents that formthe coacervate micro and/or nanodroplets and/or hydrogels may containfunctional groups that allow for attachment of targeting molecules.

The coacervate micro and/or nanodroplets and/or hydrogels can beinjectable and/or implantable, and can be in the form of a membrane,sponge, gel, solid scaffold, spun fiber, woven or unwoven mesh,nanoparticle, microparticle, or any other desirable configuration. Thecoacervate micro and/or nanodroplets and/or hydrogels can be used in avariety of biomedical applications, including tissue engineering, drugdiscovery applications, and regenerative medicine and cancer therapy.

In one example, a biodegradable coacervate hydrogel comprising suspendedchondrogenic cells, such as MSCs, and growth factor (e.g., BMP-2)encapsulated coacervate micro and/or nanodroplets can be used in amethod to promote tissue growth in a subject. One step of the method caninclude identifying a target site. The target site can comprise a tissuedefect (e.g., cartilage and/or bone defect) in which promotion of newtissue (e.g., cartilage and/or bone) is desired. The target site canalso comprise a diseased location (e.g., tumor). Methods for identifyingtissue defects and disease locations are known in the art and caninclude, for example, various imaging modalities, such as CT, MRI, andX-ray.

The tissue defect can include a defect caused by the destruction of boneor cartilage. For example, one type of cartilage defect can include ajoint surface defect. Joint surface defects can be the result of aphysical injury to one or more joints or, alternatively, a result ofgenetic or environmental factors. Most frequently, but not exclusively,such a defect will occur in the knee and will be caused by trauma,ligamentous instability, malalignment of the extremity, meniscectomy,failed ACI or mosaicplasty procedures, primary osteochondritisdessecans, osteoarthritis (early osteoarthritis or unicompartimentalosteochondral defects), or tissue removal (e.g., due to cancer).Examples of bone defects can include any structural and/or functionalskeletal abnormalities. Non-limiting examples of bone defects caninclude those associated with vertebral body or disc injury/destruction,spinal fusion, injured meniscus, avascular necrosis, cranio-facialrepair/reconstruction (including dental repair/reconstruction),osteoarthritis, osteosclerosis, osteoporosis, implant fixation, trauma,and other inheritable or acquired bone disorders and diseases.

Tissue defects can also include cartilage defects. Where a tissue defectcomprises a cartilage defect, the cartilage defect may also be referredto as an osteochondral defect when there is damage to articularcartilage and underlying (subchondral) bone. Usually, osteochondraldefects appear on specific weight-bearing spots at the ends of thethighbone, shinbone, and the back of the kneecap. Cartilage defects inthe context of the present invention should also be understood tocomprise those conditions where surgical repair of cartilage isrequired, such as cosmetic surgery (e.g., nose, ear). Thus, cartilagedefects can occur anywhere in the body where cartilage formation isdisrupted, where cartilage is damaged or non-existent due to a geneticdefect, where cartilage is important for the structure or functioning ofan organ (e.g., structures such as menisci, the ear, the nose, thelarynx, the trachea, the bronchi, structures of the heart valves, partof the costae, synchondroses, enthuses, etc.), and/or where cartilage isremoved due to cancer, for example.

After identifying a target site, such as a cranio-facial cartilagedefect of the nose, the biodegradable hydrogel can be administered tothe target site. The coacervate hydrogel can be prepared according tothe method described above.

Next, the biodegradable hydrogel may be loaded into a syringe or othersimilar device and injected or implanted into the tissue defect. Uponinjection or implantation into the tissue defect, the biodegradablecoacervate hydrogel can be formed into the shape of the tissue defectusing tactile means.

After implanting the biodegradable hydrogel into the subject, theprogenitor cells can begin to migrate from the hydrogel into the tissuedefect, express growth and/or differentiation factors, and/or promotechondroprogenitor cell expansion and differentiation. Additionally, thepresence of the biodegradable coacervate hydrogel in the tissue defectmay promote migration of endogenous cells surrounding the tissue defectinto the biodegradable hydrogel.

The following example is for the purpose of illustration only and is notintended to limit the scope of the claims, which are appended hereto.

Example 1

In this Example, we describe we describe the spontaneous formation andproperties of coacervates and/or coacervate-laden photocrosslinkedhydrogels (i.e., coacervate hydrogels) derived from the simple mixing ofOMA and GelMA in aqueous solution at a wide pH range and roomtemperature, and demonstrate that the resultant compartments can beutilized as novel platforms for localized, sustained bioactive moleculedelivery systems with the capacity for the simultaneous encapsulation ofstem cells, such as mesenchymal stem cells (MSCs), for therapeuticapplications like bone tissue engineering.

Methods Preparation of OMA/GelMA Coacervates

All macromers were dissolved separately in Dulbecco's phosphate bufferedsaline (PBS, 20 w/v %) with a photoinitiator(2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, 0.05 w/v %,Sigma). GelMA solutions were added to the OMA solutions at an equalvolume ratio, followed by mixing for 1 min, to produce the OMA/GelMAcoacervate microdroplets. The OMA/GelMA coacervate microdropletsolutions were spread on cover slips, and imaged using a fluorescencemicroscope (ECLIPSE TE 300, Nikon) equipped with a digital camera(Retiga-SRV, QImaging). Complete detailed methodology can be found inSupplementary Information.

Preparation and Characterization of Oxidized and Methacrylated Alginate(OMA)

The oxidized alginate (OA) was prepared by reacting sodium alginate(Protanal LF 20/40, 196,000 g/mol, FMC Biopolymer) with sodium periodate(Sigma) using a modification of a previously described method. Briefly,sodium alginate (10 g) was dissolved in ultrapure deionized water(diH₂O, 900 ml) overnight. Sodium periodate (1.00, 1.75 and 2.50 g) wasdissolved in 100 ml diH₂O and added to separate alginate solutions toachieve different degrees of theoretical alginate oxidation (10, 17.5and 25%, respectively) under stirring in the dark at room temperature(RT) for 24 hrs. The oxidized, methacrylated alginate (OMA) macromer wasprepared by reacting OA with 2-aminoethyl methacrylate (AEMA, Sigma). Tosynthesize OMA, 2-morpholinoethanesulfonic acid (MES, 19.52 g, Sigma)and NaCl (17.53 g) were directly added to an OA solution (1 L) and thepH was adjusted to 6.5. N-hydroxysuccinimide (NHS, 0.88, 1.47 and 2.65g; Sigma) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC, 2.92, 4.86, and 8.75 g; Sigma) (molar ratio ofNHS:EDC=1:2) were added to the mixture to activate 15, 25 and 45% of thecarboxylic acid groups of the alginate, respectively. After 5 min, AEMA(1.27, 2.11 and 3.80 g) (molar ratio of NHS:EDC:AEMA=1:2:1) was added tothe product, respectively, and the reaction was maintained in the darkat RT for 24 hrs. The reaction mixture was precipitated with theaddition of excess of acetone, dried in a fume hood overnight, andrehydrated to a 1% w/v solution in diH₂O for further purification. TheOMA was purified by dialysis against diH₂O (MWCO 3500; SpectrumLaboratories Inc.) for 3 days, treated with activated charcoal (0.5mg/100ml, 50-200 mesh, Fisher, Pittsburgh, Pa.) for 30 min, filtered(0.22 μm filter) and lyophilized To determine the levels of alginateoxidation and methacrylation, the OMAs were dissolved in deuterium oxide(D₂O, 2 w/v %), and ¹H-NMR spectra were recorded on a Varian Unity-300(300 MHz) NMR spectrometer (Varian Inc.) using3-(trimethylsilyl)propionic acid-d₄ sodium salt (0.05 w/v %) as aninternal standard. The actual oxidation and methacrylation (Table 1) ofOMAs were calculated from ¹H-NMR spectra (FIG. 1) based on a previouslydescribed method.

TABLE 1 Actual oxidation (%) and methacrylation (%) of OMAs, and actualmethacrylation (%) of GelMA Theoretical Actual Theoretical methacry-Actual methacry- oxidation lation oxidation lation Code (%)^(a) (%)^(b)(%)^(c) (%) 10OX15MA 10 15 9.05 7.54^(d) 10OX25MA 10 25 9.05 11.89^(d)10OX45MA 10 45 9.05 16.93^(d) 17.5OX15MA 17.5 15 14.22 10.23^(d)17.5OX25MA 17.5 25 14.22 14.09^(d) 17.5OX45MA 17.5 45 14.22 19.23^(d)25OX15MA 25 15 20.17 10.74^(d) 25OX25MA 25 25 20.17 16.39^(d) 25OX45MA25 45 20.17 21.38^(d) L-GelMA-A — — — 6.54^(e) L-GelMA-B — — — 7.83^(e)H-GelMA-A — — — 97.39^(e) H-GelMA-B — — — 93.23^(e) ^(a)Theoreticaloxidation of uronic acid units of alginate was calculated based on themass of alginate in 1 w/v % solution and the molecular weight of therepeat unit (M₀ = 198). ^(b)Theoretical methacrylation of the alginatecarboxylic acid reactive groups was calculated based on the mass ofalginate in 1 w/v % solution and the molecular weight of the repeat unit(M₀ = 198). ^(c)Actual alginate oxidation was calculated from ¹H-NMRdata based on a previously described method. ^(d)Actual methacrylationof OMA was calculated from ¹H-NMR data based on a previously describedmethod. ^(e)Actual methacrylation of GelMA was calculated from ¹H-NMRdata based on a previously described method.

The highly methacrylated type-A gelatin (H-GelMA-A) and type-B gelatin(H-GelMA-B) were synthesized by reaction of type-A gelatin and type-Bgelatin with methacrylic anhydride using a modification of a previouslydescribed method. Briefly, porcine skin type-A gelatin (10 g, Sigma) andbovine type-B gelatin (10 g, Sigma) were separately dissolved in 100 mLDulbecco's phosphate buffered saline (PBS, Gibco) at 60° C. and stirreduntil fully dissolved. Methacrylic anhydride (10 mL, Sigma, purity≥92%)was added at a rate of 0.5 mL/min to each gelatin solution understirring at 50° C., and the reaction was maintained in the dark at RTfor 3 hrs. Less methacrylic anhydride (0.5 mL) was used to prepare GelMAwith a lower degree of methacrylation (L-GelMA). The reaction mixturewas precipitated into excess acetone, dried in fume hood and rehydratedto a 10 w/v % solution in diH₂O. The GelMAs were purified by dialysisagainst diH₂O (MWCO 12-14 kDa) for 7 days at 40° C. to remove salts,unreacted methacrylic anhydride and byproducts, filtered (0.22 μmfilter) and lyophilized To analyze the degree of methacrylation ofGelMAs, ¹H-NMR spectra of GelMA in D₂O were recorded as described above.The actual methacrylation of GelMAs (Table 1) were calculated from the¹H-NMR spectra (FIG. 2) based on a previously described method.

Fluorescent Labeling of OMA and GelMA

Images of OMA/GelMA coacervates formed with fluorescently labeledcomponents can demonstrate the structure of the coacervatemicrodroplets. Therefore, the OMA and GelMA were labeled with differentfluorescent dyes to reveal this structure. OMA (1 g, 17.5OX15MA) wasdissolved in 100 ml MES buffer (50 mM MES, 0.5 M NaCl, and pH 6.5). NHS(0.58 g) and EDC (1.94) (molar ratio of NHS:EDC =1:2) were added to theOMA solution to activate the carboxylic acid groups of the OMA. After 5min, a blue fluorescent dye (1 mg, CF™ 350 hydrazide, Biotium) was addedto the OMA solution, and the reaction was maintained in the dark at RTfor 24 hrs. H-GelMA-A (1 g) was dissolved in 100 ml MES buffer (50 mMMES, 0.5 M NaCl, and pH 6.5), a red fluorescent dye (0.82 mg, CFTM633succinimidyl ester, Biotium) was added to the GelMA solution, and thereaction was maintained in the dark at RT for 24 hrs. The macromers wereprecipitated in excess acetone, dried in a fume hood overnight, andrehydrated to a 1% w/v solution in diH₂O for further purification. Thefluorescently labeled OMA and GelMA were purified by dialysis againstdiH₂O (MWCO 12˜14 k Da; Spectrum Laboratories Inc.) for 3 days, filtered(0.22 μm filter) and lyophilized The OMA/GelMA coacervate microdropletswere prepared, spread on a cover slip, and imaged using a fluorescencemicroscope (ECLIPSE TE 300, Nikon) equipped with a digital camera(Retiga-SRV, QImaging).

Preparation of OMA/GelMA Coacervates

All OMAs and GelMAs (20 w/v %) were dissolved separately in PBS with aphotoinitiator (2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone,0.05 w/v %, Sigma) at pH 7.4. GelMA solutions were added to the OMAsolutions at an equal volume ratio, followed by mixing for 1 mM bypipetting, to produce the OMA/GelMA coacervate microdroplets. TheOMA/GelMA coacervate microdroplet solutions were spread on cover slips,and were imaged using a microscope (ECLIPSE TE 300) equipped with adigital camera (Retiga-SRV). The coacervate microdroplet diameters weremeasured using NIH ImageJ ananlysis software (n>400 per group). Theaverage size of the coacervate microdroplets was significantly smallerfor GelMA-A compared to GelMA-B (FIG. 3D-U). As the theoretical degreeof alginate oxidation significantly increased from 10 to 17.5%, the sizeof coacervate microdroplets increased. The size of coacervatemicrodroplets also significantly increased as the theoretical degree ofalginate methacrylation increased from 15 to 25% (FIG. 3D-U).

To fabricate OMA/GelMA coacervate microdroplet-laden photocrosslinkedhydrogels, 10OX45MA, 17.5OX45MA and 25OX45MA OMA solutions were eachmixed with an equal volume of H-GelMA-B solution for 1 min by pipetting,placed between quartz (top) and glass (bottom) plates separated by 0.4mm spacers, photocrosslinked with 365 nm UV light (Omnicure® S1000, EXFOPhotonic Solution Inc.) at ˜3.0 mW/cm² for 5 min, and then imaged usinga microscope (ECLIPSE TE 300) equipped with a digital camera(Retiga-SRV).

The elastic moduli of OMA/GelMA coacervate microdroplet-ladenphotocrosslinked hydrogels were determined by performing uniaxial,unconfined constant strain rate compression tests at room temperatureusing a constant crosshead speed of 1%/sec on a mechanical testingmachine (225 lbs Actuator, TestResources) equipped with a 5 N load cell.Elastic moduli were calculated from the first non-zero linear slope ofthe stress versus strain plots over 5% strain (N=3). The compressivemoduli of coacervate microdroplet-laden photocrosslinked hydrogelsformed with H-GelMA-A (Figure S3v) were significantly higher thanH-GelMA-B (FIG. 3W) when formed with the same OMA, except for the10OX25MA and 10OX45MA compositions. The compressive moduli significantlyincreased as the degree of alginate methacrylation increased. Incontrast, as the degree of alginate oxidation increased, the compressivemoduli significantly decreased (FIG. 3V-W).

Turbidity Measurements of OA/Gelatin, OA/GelMA, OMA/Gelatin andOMA/GelMA Mixtures

All macromers were dissolved separately in PBS (20 w/v %) with aphotoinitiator (0.05 w/v %) at pH 7.4. To measure the turbidity ofalginate and gelatin mixtures, 100 μL of OA or OMA solutions were placedin wells of a 96-well plate and then mixed with 100 μL of gelatin orGelMA solutions using a pipette for 1 min. The absorbance (turbidity) at500 nm was measured before and after mixing using a microplate reader(VersaMax, Molecular Devices). The turbidity of OAs/gelatins (FIG. 4A),OMAs/gelatins (FIG. 4B), and OAs/L-GelMAs (FIG. 4C) did notsignificantly increase after mixing, indicating no coacervate formation,while OAs/H-GelMAs exhibited significant increases in turbidity (FIG.4D). Interestingly, the turbidity of OMAs/L-GelMAs exhibited greaterincreases after mixing as OMA methacrylation increased (FIG. 4E),indicating the extent of OMA/GelMA coacervate formation is also affectedby the degree of alginate methacrylation.

To evaluate the effect of the degree of gelatin methacrylation oncoacervate formation, 100 μL of OA (25% theoretical oxidation) solutionwas mixed with 100 μL of gelatin solution containing various weightfractions of H-GelMA-A in 96-well plate using a pipette for 1 min, andthen the turbidity at 500 nm was measured using a microplate reader(VersaMax).

Osteogenesis of Photoencapulated hMSCs by BMP-2 Delivery from OMA/GelMACoacervate Hydrogel

To evaluate the release kinetics of BMP-2 from photocrosslinkedOMA/GelMA coacervate hydrogels, OMA (20 w/v %, 25OX45MA) and H-GelMA-A(20 w/v %) were dissolved separately in PBS containing 0.05 w/v %photoinitiator (Sigma). BMP-2 (8 μg, R&D systems, 1 μg/μL) andI¹²⁵-labeled BMP-2 (16 μL, 250 μCi/mL, Perkin Elmer) were added to theOMA or GelMA solution. After gently mixing for 5 min, a 50 μL aliquot ofOMA solution was placed a well of a 96-well plate and then mixed with anequal volume of GelMA solution using a pipette for 1 min. The mixtureswere photocrosslinked with 365 nm UV light (Omnicure) at ˜3.0 mW/cm² for5 min to form the hydrogels. The final concentrations ofphotoencapsulated growth factors were 1μg BMP-2 and 0.5 μCi I¹²⁵-labeledBMP-2 per 100 μL hydrogel. Each photocrosslinked hydrogel was thenplaced in a 15-ml conical tube containing 5 mL PBS (pH 7.4) andincubated at 37° C. At predetermined time points over the course ofeight weeks, the supernatant was withdrawn and fresh PBS wasreplenished. Aliquots (50 μl) of supernatant were emulsified withOptiphase HighSafe-2 scintillation cocktail solution (150 μl,Perkin-Elmer) in 96-well microplates for 1 min. The radioactivity in thesupernatants was determined using a Wallac 1450 Microbeta liquidscintillation counter (Perkin-Elmer). The cumulative BMP-2 release fromthe photocrosslinked OMA/GelMA coacervate hydrogels at each time pointwas normalized as a percentage of total BMP-2 incorporated. Tofunctionalize OMA/GelMA coacervate hydrogels with heparin, methacrylatedheparin (heparin:OMA or GelMA=1:9 weight ratio) was added to OMA orGelMA solutions prior to addition of BMP-2 (8 μg) and I¹²⁵-labeled BMP-2(16 μL). After coacervate formation and subsequent photocrosslinking,the release study was performed in PBS as described above.

To isolate hMSCs, bone marrow aspirates were obtained from the posterioriliac crest of healthy donors under a protocol approved by theUniversity Hospitals of Cleveland Institutional Review Board andprocessed as previously described. Briefly, the aspirates were washedwith growth medium comprised of low-glucose Dulbecco's Modified Eagle'sMedium (DMEM-LG, Sigma) with 10% prescreened fetal bovine serum (FBS,Gibco). Mononuclear cells were isolated by centrifugation in a Percoll(Sigma) density gradient and the isolated cells were plated at 1.8×10⁵cells/cm² in DMEM-LG containing 10% FBS and 1% penicillin/streptomycin(P/S, Thermo Fisher Scientific) in an incubator at 37° C. and 5% CO₂.After 4 days of incubation, non-adherent cells were removed and adherentcell were maintained in DMEM-LG containing 10% FBS and 1 P/S with mediachanges every 3 days. After 14 days of culture, the cells were passagedat a density of 5×10³ cells/cm².

To evaluate the osteogenic efficacy of BMP-2 delivery usingphotocrosslinked OMA/GelMA coacervate microdroplet-embedded hydrogels,OMA (20 w/v %, 25OX45MA) and H-GelMA-A (20 w/v %) were dissolvedseparately in DMEM-LG containing 0.05 w/v % photoinitiator. BMP-2 (5μg/100 μl macromer solution) was added to the OMA or GelMA solution, andthen the hMSCs (passage number 3, 10×10⁶ cells/ml) were suspended in OMAsolution. After mixing the OMA solution with an equal volume of GelMAsolution, the cell/coacervate solutions were injected between two glassplates separated by 0.75 mm spacers and photocrosslinked with 365 nm UVlight at ˜3 mW/cm² for 5 min to form the hydrogel-cell constructs.Photocrosslinked hydrogel-cell construct disks were created using an 8mm diameter biopsy punch, placed in wells of 24-well tissue cultureplates with 1 ml osteogenic media [10 mM β-glycerophosphate(CalBiochem), 50 μM ascorbic acid (Wako), and 100 nM dexamethasone (MPBiomedicals)] containing 10% FBS and 1% P/S, and cultured in ahumidified incubator at 37° C. with 5% CO₂ for 112 days.

The viability of encapsulated hMSCs in the photocrosslinked OMA/GelMAcoacervate hydrogels was investigated using a Live/Dead assay comprisedof fluorescein diacetate [FDA, 1.5 mg/ml in dimethyl sulfoxide (ResearchOrganic Inc.), Sigma] and ethidium bromide (EB, 1 mg/ml in PBS, ThermoFisher Scientific). The staining solution was freshly prepared by mixing1 ml FDA solution and 0.5 ml EB solution with 0.3 ml PBS (pH 8). Atpredetermined time points, 20 μl of staining solution was added intoeach well and incubated for 3-5 min at room temperature, and thenstained hydrogel-cell constructs were imaged using a fluorescencemicroscope (ECLIPSE TE 300) equipped with a digital camera (Retiga-SRV).

To determine whether hMSCs cultured in growth factor-ladenphotocrosslinked OMA/GelMA coacervate hydrogels undergo osteogenicdifferentiation in vitro, at predetermined time points eachhydrogel-cell construct was removed from the 24-well plates, put in 1 mlALP lysis buffer and homogenized at 35,000 rpm for 30 s using a THhomogenizer (Omni International). The homogenized solutions werecentrifuged at 500 g with a Sorvall Legent RT Plus Centrifuge (ThermoFisher Scientific). The supernatants were collected for ALP, calcium andDNA analysis (N=3). For ALP measurement, supernatant (100 μl) wastreated with p-nitrophenylphosphate ALP substrate (pNPP, 100 Sigma) at37° C. for 30 min, and then 0.1 N NaOH (50 μl) was added to stop thereaction. The absorbance was measured at 405 nm using a plate reader(VersaMax). Calcium content of the encapsulated hMSCs was alsoquantified using a calcium assay kit (Pointe Scientific) according tothe manufacturer's instructions. Supernatant (4 μl) was mixed with acolor and buffer reagent mixture (250 μl) and the absorbance was read at570 nm on a microplate reader. DNA content in supernatant was alsomeasured using a Picogreen assay kit (Invitrogen) according to themanufacturer's instructions. Fluorescence intensity of thedye-conjugated DNA solution was measured using a fluorescence microplatereader (FMAX, Molecular Devices) set at 485 nm excitation and 538 nmemission. All ALP and calcium content measurements were normalized toDNA content. To visualize the calcium deposition in the hydrogel disks,hydrogel-cell constructs were fixed with 4% paraformaldehyde for 40 min,stained with Alizarin Red S (2 w/v %, pH 4.2; Sigma) for 5 min, andimaged using a digital camera (iPhone 5, Apple). Fixed hydrogel-cellconstructs were embedded in paraffin, sectioned at a thickness of 10 μm,stained with Alizarin Red S, and then imaged using a microscope (LeitzLaborlux S, Leica) equipped with a digital camera (Coolpix 995, Nikon).

RNA was isolated from hydrogel-cell constructs (N=6) with TRI Reagent(Sigma), and first-strand cDNA was synthesized with a cDNA synthesis kit(PrimeScript RT Reagent Kit with gDNA Eraser, Dakara Bio) according tothe manufacturer's instructions. qRT-PCR was performed with a SYBRPremix Ex Tag II (Tli RNase H Plus) kit (Takara Bio) according to themanufacturer's instructions. The primer sequences used for qRT-PCR arein Table 2. All reactions were run on an ABI 7500 Real-Time PCRinstrument (Applied Biosystems) for 30 sec at 95° C. followed by 40cycles of a two-step thermocycling program: 5 sec denaturing at 95° C.,34 sec annealing/extension at 60° C. Results were analyzed with SDSsoftware (Applied Biosystems) and the RQ (relative quantity) ManagerSoftware (Applied Biosystems) for automated data analysis. Relativeexpression for the target gene of interest (TGI) was normalized to GAPDHusing the delta threshold cycle (ΔCt) method. Namely, the Ct for eachgene and endogenous control GAPDH in each sample were used to create aΔCt_(TGJ) value (Ct_(TGI)−Ct_(GAPDH)). Thereafter, ΔΔCt values werecalculated by subtracting the ΔCt_(TGI) of the control (OMA/GelMA)without BMP-2 from the ΔCt_(TGI)) of experimental groups. The relativeexpression of target gene was calculated using the equation: 2^(−ΔΔCt).

TABLE 2 Primer sequences used for qRT-PCR Accession Gene DirectionPrimer sequences number GAPDH Forward ReverseGGGGCTGGCATTGCCCTCAAGGCTGGT NM_002046 GGTCCAGGGGTCT (SEQ ID NO: 1) Runx2Forward Reverse ACAGAACCACAAGTCGGTGCAATGGC NM_004348TGGTAGTGACCTGCGGA (SEQ ID NO: 2) BSP Forward ReverseACCCTAACCCTGGAGAGCCCCTTCGCC NM_004967 TTGAGATATCGGGGGCA (SEQ ID NO: 3)

Results

OMA was prepared by functionalization of alginate by both oxidation andmethacrylation (FIG. 8A), and GelMA was synthesized by methacrylatinggelatin (FIG. 8B). By mixing aqueous solutions of OMA and GelMA, iminebond-based complex coacervate microdroplets can form via Schiff basereaction between the aldehyde groups of the OMA and the amine groups ofthe GelMA (FIG. 8C). In this manner, micron-scale coacervates (FIGS.8D-F and FIGS. 3A-B) and coacervate-laden hydrogels formed byphotocrosslinking immediately after mixing (FIG. 3C) were easilygenerated. The morphological changes in the OMA/GelMA coacervatesresulting from varying the degrees of alginate oxidation andmethacrylation were first investigated. When varying the theoreticalmethacrylation level of OMA (15, 25 and 45%) while keeping a constanttheoretical oxidation level of 10% (10OX15MA, 10OX25MA, and 10OX45MA),after mixing with highly methacrylated type-A gelatin (H-GelMA-A) andtype-B gelatin (H-GelMA-B) solutions, coacervate microdroplets formedthat were not uniformly spherical in structure. In contrast, solutionsof H-GelMA mixed with OMAs of 17.5% and 25% theoretical oxidationexhibited relatively homogeneous spherical complex coacervatemicrodroplets (FIG. 3A-B). Since the crosslinking by imine bond-basedcomplexation between OMA and H-GelMA depends on the number of aldehydegroups of OMA, and 17.5OX and 25OX OMA had 1.57 and 2.23-fold higheraldehyde groups than 10OX OMAs (Table 1), respectively, it is likelythat the OMA/H-GelMA coacervate microdroplets have higher crosslinkingdensity with increasing alginate oxidation level, which could enhancethe physical stability of coacervate microdroplets.

Since turbidity is one of most important indicators to confirmingcoacervation, we evaluated turbidity before and after mixing of the twosolutions by absorbance measurement at 500 nm to determine the degreesof complex coacervate formation. Regardless of gelatin type and alginateoxidation and methacrylation level, the turbidity of all conditionssignificantly increased after mixing, indicating complex coacervateformation (FIG. 8G). As the alginate oxidation level increased, theturbidity of the OMA/H-GelMA complex coacervates also exhibited anincreasing trend, indicating more stable coacervate formation. Thisresult was well correlated with microscopic examination of themorphology of the coacervates. However, as the alginate methacrylationlevel increased, which could decrease the number of negatively chargedcarboxylic acid groups in alginate, the turbidity of the OMA/H-GelMAcoacervates showed a decreasing trend. This result indicates that theelectrostatic interactions of carboxylic acid groups of OMA and aminegroups of H-GelMA could also affect the coacervate formation of OMA andH-GelMA.

The phase separation in typical complex coacervation is primarily causedby the electrostatic interactions between oppositely chargedpolyelectrolytes such as proteins and polymers. Therefore, pH, whichinfluences the ionic strength of polyelectrolytes, plays a fundamentalrole in the formation of complexes between oppositely chargedpolyelectrolytes. Since the capacity for complex coacervate formation bythe alginate/gelatin system has exhibited a strong dependence on pH inprevious reports, the turbidity of OMA/GelMA coacervates at various pHs(2.07˜11.57) was measured to determine if there was a similar pHdependency for these functionalized polymers. Interestingly, themixtures of OMA and H-GelMA formed coacervates at a wide range of pH,demonstrating a pH independency, with higher turbidity observed at lowerpH (FIG. 8H). This result indicates that the complex coacervates weremainly formed through imine bond formation by Schiff base reaction,which takes place at a wide range of pHs, between the aldehyde groups ofOMA and the amine groups of H-GelMA as shown in FIG. 8C, while theelectrostatic interactions between the carboxylic groups of OMA and theamine groups of H-GelMA had greater affect on the complex coacervateformation at lower pH, which is in agreement with the literature.

Since the spatial distribution of polymers can significantly influencethe properties of complex coacervate systems, OMA and GelMA weremodified with water-soluble blue fluorescent CF™-350 and red fluorescentCF™-633 dyes, respectively (FIGS. 9A and B) to visualize thedistribution of the polymers in this coacervate system. As shown inFIGS. 2C-E, coacervate microdroplets were primarily composed ofH-GelMA-A (red), while OMA (blue) was mainly observed in the surroundingequilibrium phase. Furthermore, high-magnification images of anindividual coacervate microdroplet showed that H-GelMA-A was uniformlydistributed throughout the coacervate microdroplet (FIGS. 9F and H),while OMA was observed on the surface shell of coacervate microdroplet(FIGS. 9G and H). FIG. 9I schematically illustrates the proposedmechanism of OMA/GelMA coacervate formation and resultingmicrostructure. Upon mixing the two solutions (FIG. 9I-L), the GelMA canform imine bond-based covalent complexes with OMA (FIG. 9I-2) regardlessof GelMA and OMA's ionic charge. Since methacrylate groups arehydrophobic, methacrylation of alginate and gelatin could increase thehydrophobicity. Because gelatin tends to aggregate by hydrophobicinteractions, which is further enhanced by its methacrylation, GelMA canmore rapidly aggregate and form coacervate droplets within a fewseconds. Finally, OMA/GelMA complexes were located on the surface ofcoacervate microdroplets and formed an outer boundary (FIGS. 9G and2I-3), which could stabilize OMA/GelMA coacervate microdroplets aftermixing the solutions.

To further support our proposed mechanism for OMA/GelMA coacervation,which is induced by the crosslinking by imine bond formation between OMAand GelMA, and examine a potential key role of the methacrylate groupsof GelMA in coacervate formation, we evaluated coacervate formationusing oxidized alginate (OA), OMA, Gelatin-A and GelMA-A. The mixturesof OA/Gelatin-A (FIG. 10A), OMA/Gelatin-A (FIG. 10B), and OA/Gelatin-Awith low level of methacrylation (L-GelMA-A) (FIG. 10C) did not formcoacervates, but instead formed chemically crosslinked and transparenthydrogels. In contrast, mixtures of OA/Gelatin-A with high level ofmethacrylation (H-GelMA-A) (FIG. 10D), OMA/L-GelMA-A (FIG. 10E), andOMA/H-GelMA-A (FIG. 10F) formed coacervate microdroplets. These resultswere further confirmed by turbidity measurements of the mixtures (FIG.4A-E).

Based on these results, we proposed the structure of each system andconfirmed it through fluorescence microscopy. In the OA and Gelatin-Amixture without any methacrylate groups, macromers and water moleculeswere homogenously distributed in the imine bond-crosslinked hydrogels(FIG. 10G). OMA/Gelatin-A (FIG. 10H) and OA/L-GelMA-A (FIG. 10I)mixtures were also crosslinked by imine bond formation, but hydrophobicinteractions by crosslinking and methacrylation were insufficient toinduce phase separation due to the low concentration of methacrylates.Therefore, they also formed crosslinked transparent hydrogels. This wasnot the case for OA/H-GelMA-A (FIG. 10J), OMA/L-GelMA-A (FIG. 10K) andOMA/H-GelMA-A (FIG. 10L) mixtures, however, which had sufficient numberof hydrophobic domains that consisted of chemical crosslinking andmethacrylate groups, and in turn induced liquid-liquid phase separationto form coacervates. The fluorescence photomicrographs providemicrostructural data that supports the proposed mechanism for coacervateformation in this system.

Since solutions of OA with L-GelMA-A did not form a complex coacervatebut H-GelMA-A did, this indicates that methacrylate concentration in thegelatin solution plays an important role in the process. To elucidatethe relationship between coacervate formation and gelatin methacrylationlevel, turbidity measurements were taken of mixtures of OA solution withgelatin solutions containing various weight fractions of H-GelMA-A. Theturbidity of the mixture gradually increased as the H-GelMA-A contentincreased up to 10% in gelatin solution, and then rapidly increasedat >10% H-GelMA-A in the gelatin solution (FIG. 4F). This result clearlydemonstrates the dependence of OMA/GelMA coacervate formation on theconcentration of hydrophobic methacrylate groups in the gelatinsolution.

This OMA/GelMA coacervation systems exhibit microdroplet formation inOMA equilibrium phase, which can be further photocrosslinked in thepresence of low level UV light and a photoinitiator to form a hydrogel.Microspheres containing bioactive molecules can be easily incorporatedand homogenously distributed within the hydrogel through thiscoacervation approach for localized delivery and exposure of thesemolecules in a controlled and sustained manner over time to cellsincorporated in the microspheres, in the equilibrium phase of thehydrogel and/or surrounding the hydrogel (FIG. 11A). To investigatewhether incorporating human bone morphogenetic protein-2 (BMP-2) intothe GelMA solution, which mainly comprised the resulting coacervatemicrodroplets, could delay the release of the growth factor, compared toBMP-2 in the OMA solution, the release profiles of BMP-2 from twodifferent coacervate-laden hydrogel systems were measured (FIG. 11B).The growth factor release from OMA/GelMA coacervate hydrogels when BMP-2was originally in the OMA solution (red triangles) was more rapid thanrelease from coacervates formed with BMP-2 originally in the GelMAsolution (black circles). The release of BMP-2 could be further delayerby the addition of photocrosslinkable heparin into the coacervatehydrogels due to affinity binding between heparin and the growth factor(FIG. 5). The affinity interactions result from electrostaticinteractions between the negatively charged sulfate groups of heparinand the positively charged amino acid groups of the growth factor.

To investigate the effect of prolonged presentation of BMP-2 on theosteogenic differentiation of stem cells in this system, humanmesenchymal stem cells (hMSCs) were photoencapsulated in OMA/GelMAcoacervate hydrogels and cultured in osteogenic differentiation media.As shown in FIG. 11C-E and FIG. 6 high cell viability was observedthroughout all groups for 4 weeks, indicating the mixing andphotoencapsulation process, macromers, and the OMA/GelMA coacervatehydrogels themselves and their degradation products are cytocompatible.Cell/hydrogel constructs were evaluated for hMSC osteogenicdifferentiation by measuring alkaline phosphatase (ALP) activity, whichis an early osteogenic differentiation marker, determining relative mRNAexpression of Runt-related transcription factor 2 (Runx2), which is oneof the earlier and most specific osteogenic differentiation makers, andbone sialoprotein (BSP), which is a later osteogenic differentiationmarker, staining for calcium using Alizarin red S, and quantifyingcalcium deposition. Compared to the OMA/GelMA group without BMP-2, theALP activity of photoencapsulated hMSCs could more rapidly increase byBMP-2 delivery from the coacervate or equilibrium phase by day 28, andthen gradually decreased (FIG. 11F). When the BMP-2 was delivered fromthe coacervate phase, photoencapsulated hMSCs showed significantlyhigher ALP activity at day 28, compared to BMP-2 delivered formequilibrium phase, and maximal ALP activity over the duration of theexperiment was quantified for both of these conditions at this timepoint. A quantitative analysis of mRNA expression levels of Runx2 andBSP were evaluated by real-time quantitative reversetranscription-polymerase chain reaction (qRT-PCR) (FIGS. 11G-H).Compared to the control group without BMP-2, hMSCs expressedsignificantly higher Runx2 by BMP-2 delivery from coacervate orequilibrium phase at day 14 (FIG. 11G). In addition, Runx2 expressionlevel of hMSCs in the OMA/BMP-2 in GelMA group was also significantlyhigher than that of the control group by day 28. Photoencapsulated hMSCsexpressed significantly higher BSP when BMP-2 was delivered from thecoacervate hydrogels compared to the controls at day 28, while there wasno significant difference among any groups at day 14 (FIG. 11H).

Since mineralization is the ultimate indicator of stem cell osteogenicdifferentiation, the calcium deposition in the hMSC/hydrogel constructswas then visualized and quantified by Alizarin red staining and acalcium assay, respectively. Compared to the control group, more intenseAlizarin red staining was observed in the BMP-2 delivery groups at days28 and 56 (FIGS. 11I-J). Moreover, the intensity of the staining signalwas greatest when the BMP-2 was loaded in the coacervate phase. As shownin FIG. 11K, similar to the Alizarin red staining results, calciumdeposition was significantly higher up to day 112 in coacervatehydrogels groups delivering BMP-2 compared to the control group, withthe highest calcium deposition in the OMA/BMP-2 in GelMA hydrogelslikely due to BMP-2 presentation in the hydrogels for a longer period oftime. As shown in FIG. 7, heparin modification of coacervate hydrogelsfurther enhanced the calcium deposition in the coacervate hydrogels.These results demonstrate that long-term presentation of bioactive BMP-2in the coacervate hydrogels enhances osteogenic differentiation ofphotoencapsulated stem cells and bone-related mineralization of theextracellular environment.

Micro- or nanoparticle-incorporated hydrogels have been widely studiedto achieve sustained localized delivery of bioactive molecules fortissue engineering applications such as regenerating bone or cartilage.Localized and controlled spatial and temporal presentation of thesebioactive molecule have been demonstrated to be valuable in regulatingencapsulated cell behavior in such tissue engineering strategies.However, in these systems, it can be technically challenging tofabricate the micro- or nanoparticles with encapsulated bioactivemolecules without loss of their bioactivity due to use of organicsolvents, high temperatures and/or shear stress. In this example, asystem has been successfully engineered for the spontaneous formation ofcoacervates and/or coacervate-laden photocrosslinked hydrogels derivedfrom the simple mixing of OMA and GelMA in aqueous solutions atphysiological conditions for long-term localized delivery of growthfactor. We demonstrated that the resultant compartments could beutilized as a novel platform for localized, sustained bioactive moleculedelivery to encapsulated stem cells for therapeutic applications likebone tissue engineering. In addition to delivery of a single growthfactor, particle-based systems have also been implemented to presentmultiple growth factors, such as BMP-2 and BMP-7, BMP-2 and insulin-likegrowth factor, vascular endothelial growth factor (VEGF) and BMP-2, andVEGF and platelet-derived growth factor (PDGF), to drive and enhancebiologic processes such as osteogenesis and angiogenesis. The coacervatesystem presented here exhibited differential release profiles of asingle growth factor depending on whether heparin was used in the systemand whether the growth factor was within the coacervate microdroplets orthe surrounding equilibrium phase. Thus, the OMA/GelMA coacervatemicrodroplet-embedded hydrogel platform could be utilized for theregulated spatiotemporal presentation of multiple growth factors fromthe same system, which could synergistically enhance tissueregeneration.

In summary, the spontaneous formation of coacervate microdroplets and/orcoacervate-laden photocrosslinked hydrogels derived from the simplemixing of photocrosslinkable OMA and GelMA over a wide pH range at roomtemperature has been demonstrated. This system enables simultaneouscreation of drug-laden microdroplets and encapsulation of stem cells inphotopolymerized coacervate hydrogels under physiological conditions andcan be utilized as a novel platform for in situ formation of localized,sustained bioactive molecule delivery to encapsulated stem cells fortherapeutic applications.

From the above description of the invention, those skilled in the artwill perceive improvements, changes and modifications. Suchimprovements, changes and modifications within the skill of the art areintended to be covered by the appended claims. All references,publications, and patents cited in the present application are hereinincorporated by reference in their entirety.

1-20. (canceled)
 21. A method for promoting tissue growth in a subjectcomprising: administering a coacervate hydrogel to a target site in thesubject, the coacervate hydrogel comprising: crosslinked oxidizedalginate and methacrylated gelatin that form a hydrogel matrix and aplurality of coacervate microdroplets and/or nanodroplets suspended inthe matrix, wherein the oxidized alginate has an oxidation percentage ofat least 10% of uronic acid units of alginate and the methacrylatedgelatin has a methacrylation percentage of about 10% to about 99% ofamine groups of gelatin.
 22. The method of claim 21, wherein at leastone bioactive agent is incorporated in the coacervate microdropletsand/or nanodroplets and/or matrix.
 23. The method of claim 21, whereinthe oxidized alginate has an oxidation percentage of up to 50% of uronicacid units of alginate.
 24. The method of claim 21, wherein the oxidizedalginate is methacrylated and has a methacrylation percentage up to 45%of alginate carboxylic acid reactive groups.
 25. The method of claim 22,wherein the hydrogel matrix includes a plurality of cells and theplurality of coacervate microdroplets and/or nanodroplets providecontrolled release of the bioactive agent to the plurality of cells. 26.The method of claim 25, wherein the bioactive agent comprises BMP-2 andthe cells comprise hMSCs.
 27. The method of claim 21, wherein thecoacervate hydrogel is administered to a tissue defect.
 28. The methodof claim 27, wherein the tissue defect is a bone and/or cartilagedefect.
 29. A method for promoting tissue growth in a subjectcomprising: administering a coacervate hydrogel to a target site in thesubject, the coacervate hydrogel comprising: a crosslinked oxidizedmethacrylated alginate and a methacrylated gelatin that form a hydrogelmatrix and a plurality of coacervate microdroplets and/or nanodroplets,wherein the oxidized methacrylated alginate has an oxidation percentageof 10% up to 50% of uronic acid units and a methacrylation percentage upto 45% of carboxylic acid reactive groups of alginate, the methacrylatedgelatin has a methacrylation percentage of at least about 10% to about99% of amine groups of alginate.
 30. The method of claim 28, wherein atleast one bioactive agent is incorporated in the microdroplets and/ornanodroplets and/or matrix.
 31. The method of claim 30, wherein thehydrogel matrix includes a plurality of cells and the plurality ofcoacervate microdroplets and/or nanodroplets provide controlled releaseof the bioactive agent to the plurality of cells.
 32. The method ofclaim 31, wherein the bioactive agent comprises BMP-2 and the cellscomprise hMSCs.
 33. The method of claim 29, wherein the coacervatehydrogel is administered to a tissue defect.
 34. The method of claim 33,wherein the tissue defect is a bone and/or cartilage defect.